この作品は、JEL、およびHA、FCA、とJDCのトロントフェローシップの大学にオンタリオ州のHIV治療ネットワーク研究オペレーティング·グラント（ROG-G645）と保健研究の新しい奨励賞のカナダ研究所（MSH-113554）でサポートされていました。著者らは、細胞を提供するためのスクリップス研究所（カリフォルニア州ラホーヤ）、エボラウイルスGPの発現ベクターと一般的なアドバイスでマーニーフスコ、Dafnaアベルソン博士とエリカOllmannサファイアに感謝します。

<ol>
	<li>Celik, E., Calik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+by+yeast+cells.">Production of recombinant proteins by yeast cells.</a> Biotechnol. Adv. , (2011).</li><li>Yokoyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+expression+systems+for+structural+genomics+and+proteomics.">Protein expression systems for structural genomics and proteomics.</a> Curr. Opin. Chem. Biol. 7, 39-43 (2003).</li><li>Nettleship, J. E., Assenberg, R., Diprose, J. M., Rahman-Huq, N., Owens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+production+of+proteins+in+insect+and+mammalian+cells+for+structural+biology.">Recent advances in the production of proteins in insect and mammalian cells for structural biology.</a> J. Struct. Biol. 172, 55-65 (2010).</li><li>Carlson, E. D., Gan, R., Hodgman, C. E., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+protein+synthesis:+Applications+come+of+age.">Cell-free protein synthesis: Applications come of age.</a> Biotechnol. Adv. , (2011).</li><li>Lee, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+Ebola+virus+glycoprotein+bound+to+an+antibody+from+a+human+survivor.">Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.</a> Nature. 454, 177-182 (2008).</li><li>Bowden, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+Nipah+and+Hendra+virus+attachment+to+their+cell-surface+receptor+ephrin-B2.">Structural basis of Nipah and Hendra virus attachment to their cell-surface receptor ephrin-B2.</a> Nat. Struct. Mol. Biol. 15, 567-572 (2008).</li><li>Evans, M. J., Hartman, S. L., Wolff, D. W., Rollins, S. A., Squinto, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+expression+of+an+anti-human+C5+chimeric+Fab+utilizing+a+vector+that+replicates+in+COS+and+293+cells.">Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.</a> J. Immunol. Methods. 184, 123-138 (1995).</li><li>Ye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+protein+expression+in+scalable+CHO+transient+transfection.">High-level protein expression in scalable CHO transient transfection.</a> Biotechnol. Bioeng. 103, 542-551 (2009).</li><li>Wurm, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+protein+therapeutics+in+cultivated+mammalian+cells.">Production of recombinant protein therapeutics in cultivated mammalian cells.</a> Nat Biotechnol. 22, 1393-1398 (2004).</li><li>Lee, J. E., Fusco, M. H., Hessell, A. J., Burton, D. R., Saphire, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+and+tactics+used+in+determining+the+structure+of+trimeric,+prefusion+Ebola+virus+GP.">Techniques and tactics used in determining the structure of trimeric, prefusion Ebola virus GP.</a> Acta Cryst. 65, 1162-1180 (2009).</li><li>Aricescu, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+expression:+developments+for+structural+proteomics.">Eukaryotic expression: developments for structural proteomics.</a> Acta. Crystallogr. D Biol. Crystallogr. 62, 1114-1124 (2006).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta. Crystallogr. D Biol. Crystallogr. D62, 1243-1250 (2006).</li><li>Chang, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+structural+genomics:+solving+the+glycosylation+problem.">Glycoprotein structural genomics: solving the glycosylation problem.</a> Structure. 15, 267-273 (2007).</li><li>Lee, J. E., Fusco, M. H., Saphire, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+platform+for+screening+expression+and+crystallization+of+glycoproteins+produced+in+human+cells.">An efficient platform for screening expression and crystallization of glycoproteins produced in human cells.</a> Nature Protocols. 4, 592-604 (2009).</li><li>Nettleship, J. E., Rahman-Huq, N., Owens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+production+of+glycoproteins+by+transient+expression+in+Mammalian+cells.">The production of glycoproteins by transient expression in Mammalian cells.</a> Methods Mol. Biol. 498, 245-263 (2009).</li><li>Neil, S. J., Zang, T., Bieniasz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetherin+inhibits+retrovirus+release+and+is+antagonized+by+HIV-1+Vpu.">Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu.</a> Nature. 451, 425-430 (2008).</li><li>Nakamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterodimerization+of+the+IL-2+receptor+beta-+and+gamma-chain+cytoplasmic+domains+is+required+for+signalling.">Heterodimerization of the IL-2 receptor beta- and gamma-chain cytoplasmic domains is required for signalling.</a> Nature. 369, 330-333 (1994).</li><li>Wang, X., Rickert, M., Garcia, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+quaternary+complex+of+interleukin-2+with+its+alpha,+beta,+and+gammac+receptors.">Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors.</a> Science. 310, 1159-1163 (2005).</li></ol>

利害の衝突が宣言されません。

10層の細胞の工場は、蛋白質のミリグラム量の生産のための効果的な容器です。このようなローラーボトル、振盪フラスコまたはスピナーフラスコのような他の伝統的な船、上のセルファクトリを使用する主な利点は、追加の実験装置を購入する必要がないということです。標準的なCO 2インキュベーター（〜6.0立方フィート）は、簡単に4つの10層の細胞工場（ 図2）を収...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td>コメント</td></tr><tr><td>アルカリホスファターゼ（BCIP / NBT）液体基質溶液</td><td>シグマ</td><td> B6404 </td><td></td></tr><tr><td>抗生物質/抗真菌剤、100X </td><td>インビトロジェン</td><td> 15240062 </td><td></td></tr><tr><td>抗HAアフィニティマトリックス、クローン3F10 </td><td>ロシュ社</td><td> 1815016 </td><td></td></tr><tr><td>抗HAマウスmAb、クローン16B12 </td><td> Covance </td><td> MMS-101P </td><td></td></tr><tr><td>細胞培養フラスコ、処理されたT75 cm 2の組織培養</td><td>コー​​ニング</td><td> 430641 </td><td></td></tr><tr><td>細胞培養フラスコ、処理されたT225 cm 2の組織培養</td><td>コー​​ニング</td><td> 431082 </td><td></td></tr><tr><td>治療-1,6 - ウェル組織培養細胞培養プレート</td><td>コー​​ニング</td><td> 3516 </td><td></td></tr><tr><td>セルファクトリ、10層のCellSTACK </td><td>コー​​ニング</td><td> 3312 </td><td></td></tr><tr><td>セントラオメガ5Kカセット</td><td>ポール</td><td> OS005C12 </td><td></td></tr><tr><td>セントラオメガ30Kカセット</td><td>ポール</td><td> OS030C12 </td><td></td></tr><tr><td>クロマトグラフィーガラスカラム、1.0×10センチメートル</td><td> Kontes </td><td> 4204001010 </td><td></td></tr><tr><td>シプロフロキサシン</td><td>シグマ</td><td> 17850 </td><td></td></tr><tr><td> CO 2 </td><td></td><td></td><td></td></tr><tr><td>ダルベッコ改変イーグル培地（DMEM） </td><td>シグマ</td><td> D5796 </td><td></td></tr><tr><td>ウシ胎児血清（FBS）、不活化熱</td><td>インビトロジェン</td><td> 12484-028 </td><td></td></tr><tr><td>トランスフェHDトランスフェクション試薬</td><td>プロメガ</td><td> 4709691001 </td><td></td></tr><tr><td> GeneJuiceトランスフェクション試薬</td><td> EMD /メルク</td><td> 70967から6 </td><td></td></tr><tr><td>グリシン</td><td>シグマ</td><td> G8898 </td><td></td></tr><tr><td>ヤギ抗マウスIgG F（ab &#39;）2をアルカリ性</td><td>サーモフィッシャーサイエンティフィック</td><td> 31324 </td><td></td></tr><tr><td>ホスファターゼ標識抗体</td><td></td><td></td><td></td></tr><tr><td>ヘマグルチニン（HA）ペプチド、100 mgの</td><td> Genscript </td><td>カスタム合成</td><td></td></tr><tr><td> （シーケンス：YPYDVPDYA 95％純度） </td><td></td><td></td><td></td></tr><tr><td> HEK 293T細胞</td><td> ATCC </td><td> CRL-11268 </td><td></td></tr><tr><td> HouseholD漂白剤（4％w / vの次亜塩素酸ナトリウム） </td><td>様々なブランドが利用可能です</td><td></td><td></td></tr><tr><td>イモビロン-P PVDF膜</td><td>ミリポア</td><td> IPVH07850 </td><td></td></tr><tr><td>ミニプレップのプラスミド精製キット、PureLinkクイック</td><td>インビトロジェン</td><td> K2100-11 </td><td></td></tr><tr><td> MaxiPrepプラスミド精製キットPureLink HiPure </td><td>インビトロジェン</td><td> K2100-07 </td><td></td></tr><tr><td> NaN 3を </td><td>シグマ</td><td> S8032 </td><td></td></tr><tr><td> pDISPLAY発現ベクター</td><td>インビトロジェン</td><td> V660-20 </td><td></td></tr><tr><td> 100Xペニシリン/ストレプトマイシン（ペン/連鎖球菌） </td><td>インビトロジェン</td><td> 15140-122 </td><td></td></tr><tr><td>リン酸緩衝生理食塩水（PBS）、滅菌1X </td><td>シグマ</td><td> D8537 </td><td></td></tr><tr><td>ポリエチレンイミン（PEI）、線形25kDaの</td><td> Polyscience </td><td> 23966 </td><td></td></tr><tr><td>ミルク乾燥粉末をすくい取る</td><td>カーネーション</td><td></td><td></td></tr><tr><td> Stericup-GP PES真空ろ過装置、 </td><td>ミリポア</td><td> SCGPU05RE </td><td></td></tr><tr><td> 0.22μmの、500mlの容量</td><td></td><td></td><td></td></tr><tr><td>トリパンブルー</td><td>インビトロジェン</td><td> 15250061 </td><td></td></tr><tr><td>トリプシン-EDTA、0.05％（w / v）の</td><td>インビトロジェン</td><td> 25300-054 </td><td></td></tr><tr><td> Tween-20を</td><td>シグマ</td><td> P7949 </td><td></td></tr><tr><td>バルプロ酸</td><td>シグマ</td><td> P4543 </td><td></td></tr><tr><td>タンジェンシャルフローシステムセントラ</td><td>ポール</td><td></td><td></td></tr><tr><td> CO 2の加湿インキュベーター、標準6.0立方。フィート</td><td>様々なブランドが利用可能です</td><td></td><td></td></tr><tr><td>電気泳動およびトランスファーユニット</td><td>様々なブランドが利用可能です</td><td></td><td></td></tr><tr><td>インキュベーター、37°C </td><td>様々なブランドが利用可能です</td><td></td><td></td></tr></tbody></table>

a convenient and general expression platform for the production of secreted proteins from human cells

細菌における組換えタンパク質の発現、一般的にE.大腸菌は 、蛋白質のミリグラム量の発現のための最も成功した戦略であった。しかし、原核生物宿主は、多くの人間は、ウイルスまたは真核生物の外国の高分子の毒性に起因するタンパク質、タンパク質の折り畳み機構の違い、または特定のCO-または細菌の翻訳後修飾の不足のための発現に応じてではありません。酵母（P.パストリスもしくはS.セレビシエ ）1,2、バキュロウイルス感染昆虫（S. frugiperdaまたは T NI）セル3、 および in vitro翻訳システムにおいて 、無細胞に基づく発現系は、2,4は正常にするために使用されている哺乳動物のタンパク質を生成します。直感的に、ベストマッチは、適切な翻訳後修飾を含む組換えタンパク質の生産を確保するために哺乳動物宿主を使用することです。哺乳動物細胞株の数（ヒト胚キッドネイ（HEK）S V40 larget T-抗原（COS）、チャイニーズハムスター卵巣（CHO）などを運ぶO riginで293、C V-1細胞）は、正常ヒトタンパク質の数ミリグラムの量を過剰発現するように利用されてきた5-9しかし、哺乳動物細胞を使用することの利点は、多くの場合、安定発現細胞株を開発するコストが高く、特殊な実験装置の要件は、低タンパク質の収量と、長い時間によって打ち消されています。コストを低く維持しながら、より高速な収率と生産するタンパク質を増加し、多くの学術的、商業的な研究所の主要な要因である。 ここでは、時間とコスト効率に優れ、接着HEK 293T細胞から分泌されるヒトタンパ​​ク質の発現のための2つの部分から成る手順を説明します。このシステムは、構造生物物理学的および生化学的研究のために機能的な蛋白質のミリグラム量にグラムを生産することが可能です。最初の部分は、目的の遺伝子の複数の構文が生成さです。並行して一時的に小さなスケールの付着HEK 293T細胞にトランスフェクションのd。細胞培養培地中に分泌された組換えタンパク質の検出および分析は、ベクトルでエンコードされたタンパク質精製タグに対する市販の抗体を用いたウェスタンブロット分析によって実行されます。その後、大規模なタンパク質の生産に適したコンストラクトを一過性に10層の細胞工場でポリエチレンイミン（PEI）を用いてトランスフェクトされる。馴化培地リットルボリュームに分泌されるタンパク質は、抗-HAアフィニティークロマトグラフィーによって精製してタンジェンシャルフローろ過を使用して、管理しやすい量に集中している。このプラットフォームのユーティリティは、サイトカイン、サイトカイン受容体、細胞表面受容体、本質的な制限要因、およびウイルスの糖蛋白質のミリグラム量を表現する能力によって証明されています。このメソッドは、成功した三量体エボラウイルス糖タンパク質5,10の構造決定で使用されています。 2インキュベーター以外の追加の機器は、必要ありません。この手順は、急速にそのようなタンパク質複合体、抗原と抗体、ワクチン、または困難な細胞株の形質導入のためのアデノウイルスまたはレンチウイルスの生産のためのウイルス様粒子の生産の共発現として、より複雑なシステムに拡張することができます。

Watch this Scientific Journal Video about A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells at JoVE.com

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

ポストヒトゲノム時代において、ネイティブコンフォメーションにおける組換えタンパク質の可用性は、構造、機能および治療の研究開発に不可欠である。ここでは、テストと組換えタンパク質のさまざまなを生成するために使用することができるヒト胚性腎臓293T細胞における大規模タンパク質発現系を記述します。

ヒト細胞から分泌タンパク質の生産のための便利で一般式プラットフォーム

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

a-convenient-general-expression-platform-for-production-secreted

Research

JoVE Journal

Biology

21.1K ビュー.  University of Toronto. ポストヒトゲノム時代において、ネイティブコンフォメーションにおける組換えタンパク質の可用性は、構造、機能および治療の研究開発に不可欠である。ここでは、テストと組換えタンパク質のさまざまなを生成するために使用することができるヒト胚性腎臓293T細胞における大規模タンパク質発現系を記述します。

ビデオ: A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

ヒト胚性幹（ES）細胞の増殖

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

組換えタンパク質の高レベルの一過性発現のための植物の効率的なアグロ

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

質量分析のためのペプチドに組織および消化から細胞外マトリックスタンパク質の濃縮

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

ヒト細胞から分泌タンパク質の生産のための便利で一般式プラットフォーム

要約

さらに動画を探す

この動画の章

ヒト胚性幹（ES）細胞の増殖

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

EMBLでMultiBacタンパク質複合体生産プラットフォーム

組換えタンパク質の高レベルの一過性発現のための植物の効率的なアグロ

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで

質量分析のためのペプチドに組織および消化から細胞外マトリックスタンパク質の濃縮

懸濁液中のHEK293細胞の一過性トランスフェクションによる組換えヒトタンパク質のハイ・イールド式

ヒト細胞から分泌タンパク質の生産のための便利で一般式プラットフォーム

要約

さらに動画を探す

この動画の章

ヒト胚性幹（ES）細胞の増殖

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

EMBLでMultiBacタンパク質複合体生産プラットフォーム

組換えタンパク質の高レベルの一過性発現のための植物の効率的なアグロ

生物物理学研究のための昆虫細胞からの効率的な生産と組換えマウスKindlin-3の精製

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで

質量分析のためのペプチドに組織および消化から細胞外マトリックスタンパク質の濃縮

懸濁液中のHEK293細胞の一過性トランスフェクションによる組換えヒトタンパ​​ク質のハイ・イールド式

懸濁液中のHEK293細胞の一過性トランスフェクションによる組換えヒトタンパク質のハイ・イールド式